Primary hepatocyte isolation and culture

Hepatocytes were isolated from non-treated animals and rodent models of BEC/HPC activation using a modified perfusion technique and cultured⁴³. In brief, Liver Perfusion Medium (Thermo Fisher Scientific, 17701038) for 5 min followed by Liver Digest Medium (Thermo Fisher Scientific, 17703034) for 10 min were used for perfusion. The liver was subsequently excised, and the capsule disrupted to yield a cell suspension in Liver Perfusion Medium, which was passed through a Falcon 100 μm cell strainer (Corning, 352360). Hepatocytes were then pelleted by centrifugation at 135 G for 1 min, separated from a non-parenchymal cell-rich fraction, and resuspended in Williams E Medium (Thermo Fisher Scientific, 12551032) with 5% FBS. Cells were underlayered with a discontinuous Percoll gradient (1.06, 1.08 and 1.12 mg/ml Percoll in PBS) and spun at 750 G for 20 min at 20 °C. Cells collected between the 1.08 and 1.12 mg/ml Percoll layers were then harvested and resuspended in Williams medium.